Substrate Peptide Interactions with the ClpA Hexamer. (GP, AG, MRM, SKS, JAR) ClpA is an ATP-dependent chaperone that forms a hexameric ring that alone has an unfoldase activity in vitro. Two of these rings flank the ClpP tetradecamer-forming ClpAP protease. In this complex, ClpA assists the proteolytic core by binding, unfolding, and translocating substrates to the proteolytic chamber. Thermodynamic parameters of ClpA-peptide interactions have been determined by isothermal titration calorimetry (ITC) using a known 11-amino acid substrate ANDENYALAA (SsrA) and alpha-SsrA and by fluorescence titrations using dansylated SsrA peptide (DNS-SsrA). The DNS-SsrA has been found to be competitively displaced by other substrates of ClpA. Peptides containing the sequences of different parts of known substrates of ClpA and ClpX were used for competition with DNS-SsrA in fluorescent studies. This technique allows identification of specific motifs responsible for recognition by ClpA. Fluorescence methods have also been used to study the effect of adaptor proteins, such as ClpS, on the ClpA-peptide interactions. The fact that peptide sequences from different substrates compete with DNS-SsrA suggests that they occupy or overlap the same binding site of the ClpA hexameric ring. Conformational Stability and Domain Coupling in Glucose/Galactose-Binding Protein. (GP, AG, SA, MS, MR) The monomeric D-Glucose/D-Galactose-Binding Protein (GGBP) from Escherichia coli is a periplasmic protein that serves as a high-affinity receptor for the active transport and chemotaxis towards both sugars. Tight binding of sugars by the binding site located in the cleft between the two domains of the GGBP is achieved by the formation of hydrogen bonds with the ligand. Glucose and galactose binding induces a hinge motion between the two domains of the GGBP protein. GGBP become an important model for reagentless glucose sensor development due to the opportunity to measure sugar concentration by monitoring induced protein conformational change. The effect of D-glucose binding on the thermal unfolding of the GGBP protein at pH 7.0 has been measured by differential scanning calorimetry (DSC), far-UV circular dichroism, and intrinsic Trp fluorescence. All three techniques reveal reversible, thermal transitions and a midpoint temperature (Tm) increase from 50 to 63 C produced by 10 mM D-glucose. Both in the absence and presence of glucose, a single asymmetric endotherm for GGBP is observed in DSC, although each endotherm consists of two transitions about 4 C apart in Tm values. In the absence of glucose, the protein unfolding is best described by two non-ideal transitions, suggesting the presence of unfolding intermediates. In the presence of D-glucose, protein unfolding is more cooperative than in the absence of the ligand, and the experimental data are best fitted to a model that assumes two ideal (two-state) sequential transitions. Thus, D-glucose binding changes the character of the GGBP protein folding/unfolding by linking the two domains such that protein unfolding becomes a cooperative, two two-state process. An association constant of 5.9 E6 1/M at 63 C for glucose binding is estimated from DSC results. The domain with the lower stability in DSC measurements has been identified as the C-terminal domain of GGBP from thermally induced Trp fluorescence changes.